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J. Am. Chem. Soc. 1996, 118, 3972-3973
Direct Observation by FTIR Spectroscopy of the Ferrous Heme-NO+ Intermediate in Reduction of Nitrite by a Dissimilatory Heme cd1 Nitrite Reductase Yaning Wang† and Bruce A. Averill* E. C. Slater Institute, UniVersity of Amsterdam Plantage Muidergracht 12, 1018 TV Amsterdam, Netherlands ReceiVed NoVember 17, 1995 Denitrification is the dissimilatory reduction of nitrogenoxygen species by certain bacteria.1-3 It constitutes a key part of the global nitrogen cycle, in that denitrification is responsible for evolution of N2(g) from the biosphere and geosphere to replenish the atmosphere. In addition, denitrification causes a substantial reduction in crop yields, since up to 25-30% of added nitrogen fertilizer can be transformed to N2 and N2O by soil microorganisms. One of the products of denitrification, N2O, is a “greenhouse gas” that has also been linked to ozone destruction in the stratosphere.4 In most, if not all, organisms, denitrification occurs in four steps via the sequence:
NO3- f NO2- f NO f N2O f N2 The step involving the reduction of nitrite has been the focus of substantial attention and considerable controversy,2,3 because it represents the branch point from assimilatory nitrate reduction and a possible point of attack for development of agricultural chemicals that might selectively inhibit denitrification. Two types of nitrite reductase are known from denitrifying bacteria.5 One is a copper-containing enzyme that exists in most cases as a trimer; each monomer contains both an unusual type 1 Cu site and a type 2 Cu site that is the site of NO2reduction.6,7 The second and more commonly encountered in nature is heme-containing enzymes, the cd1 NiR’s.5 All examples characterized to date are dimers, with each monomer containing both a heme c and a heme d1 (dioxoisobacteriochlorin) chromophore.9 The roles of the two heme centers in catalysis remain unresolved, although the heme d1 is presumed to be the site at which nitrite is reduced. Substantial evidence has been adduced for the existence of an electrophilic hemenitrosyl intermediate in the reduction of nitrite (formulated as either Fe2+-NO+ or Fe3+-NO), based primarily on isotope exchange and trapping experiments.10,11 Its formation from nitrite is, however, too fast to be detected even by stoppedflow studies.12 We report herein the results of FTIR studies of the heme cd1 nitrite reductase from Pseudomonas stutzeri JM300 in which we have been able to detect an Fe2+-NO+ species formed by reaction of NO product with the oxidized enzyme; * To whom correspondence should be addressed: 31-20-525-5045 (Fax); 31-20-525-5124 (phone);
[email protected] (e-mail). † Research performed at University of Virginia, Charlottesville, VA 22901. (1) Payne, W. J. Denitrification; John Wiley & Sons: New York, 1981. (2) Ye, R. W.; Averill, B. A.; Tiedje, J. M. Appl. EnViron. Microbiol. 1994, 60, 1053-1058. (3) Zumft, W. G. Arch. Microbiol. 1993, 160, 253-264. (4) Rasmussen, R. A.; Khalil, M. A. K. Science 1986, 232, 1623-1624. (5) Hochstein, L. I.; Tomlinson, G. A. Ann. ReV. Microbiol. 1988, 42, 231-261. (6) Godden, J. W.; Turley, S.; Teller, D. C.; Adman, E. T.; Liu, M. Y.; Payne, W. J.; LeGall, J. Science 1991, 253, 438-442. (7) Libby, E.; Averill, B. A. Biochem. Biophys. Res. Commun. 1992, 187, 1529-1535. (8) Coyne, M. S.; Arunakumari, A.; Averill, B. A.; Tiedje, J. M. Appl. EnViron. Microbiol. 1989, 55, 2924-2931. (9) Chang, C. K. J. Biol. Chem. 1986, 261, 8593-8596. (10) Kim, C.-H.; Hollocher, T. C. J. Biol. Chem. 1984, 259, 2092-2099. (11) Garber, E. A. E.; Hollocher, T. C. J. Biol. Chem. 1982, 257, 80918097. (12) Silvestrini. M. C.; Tordi, M. G.; Musci, G.; Brunori, M. J. Biol. Chem. 1990, 265, 11783-11787.
0002-7863/96/1518-3972$12.00/0
in addition, we report FTIR spectra of the NO complex of methemoglobin. Infrared spectroscopy has proven to be a sensitive technique for the direct observation of certain ligands bound to metalloproteins and for probing the local environment of the ligand binding site.13,14 The ligands most commonly used in such studies are CO, CN-, and N3-.15-18 In contrast, NO has been seldom studied in this regard, because the N-O stretch for NO itself and NO bound to most metal centers lies in the same spectral region as the strong protein amide I band. Complexes in which NO is bound to more oxidized metal centers should have appreciable NO+ character and should exhibit N-O stretches at higher frequencies, where there is less interference from the protein background. Unfortunately, such complexes tend to be less stable. The only such reports of which we are aware are studies of nitrosylhemoglobin, Hb-NO, for which νNO values of 1587 and 1615 cm-1 were reported for 15NO and 14NO, respectively.19 In these studies, NO complex of ferric horseradish peroxidase (HRP) was reported to give a νNO at 1865 cm-1 for 15NO19a and the NO complex of metHb was reported to give a νNO at 1925 cm-1 for 14NO,19b but no spectra were shown. Consequently, we began by examining the FTIR spectra of concentrated samples of metHb.20 Optical spectra of metHb treated with 1 atm NO (not shown) show that a new species with λmax at 533 and 566 nm is formed rapidly (
